Use of vegf and homologues to treat neuron disorders

ABSTRACT

The present invention relates to neurological and physiological dysfunction associated with neuron disorders. In (particular, the invention relates to the involvement of vascular endothelial growth factor (VEGF) and homologues in the aetiology of motor neuron disorders. The invention further concerns a novel, mutant transgenic mouse (VEGF m/m ) with a homozygous deletion in the hypoxia responsive element (HRE) of the VEGF promoter which alters the hypoxic upregulation of VEGF. These mice suffer severe adult onset muscle weakness due to progressive spinal motor neuron degeneration which is reminiscent of amyotrophic lateral sclerosis (ALS)—a fatal disorder with unknown aetiology. Furthermore, the neuropathy of these mice is not caused by vascular defects, but is due to defective VEGF-mediated survival signals to motor neurons. The present invention relates in particular to the isoform VEGF 165  which stimulates survival of motor neurons via binding to neuropilin-1, a receptor known to bind semaphorin-3A which is implicated in axon retraction and neuronal death, and the VEGF Receptor-2. The present invention thus relates to the usage of VEGF, in particular VEGF 165 , for the treatment of neuron disorders and relates, in addition, to the usage of polymorphisms in the VEGF promotor for diagnosing the latter disorders.

FIELD OF THE INVENTION

[0001] The present invention relates to neurological and physiologicaldysfunction associated with neuron disorders. In particular, theinvention relates to the involvement of vascular endothelial growthfactor (VEGF) and homologues in the aetiology of motor neuron disorders.The invention further concerns a novel, mutant transgenic mouse(VEGF^(m/m)) with a homozygous deletion in the hypoxia responsiveelement (HRE) of the VEGF promoter which alters the hypoxic upregulationof VEGF. These mice suffer severe adult onset muscle weakness due toprogressive spinal motor neuron degeneration which is reminiscent ofamyotrophic lateral sclerosis (ALS)—a fatal disorder with unknownaetiology. Furthermore, the neuropathy of these mice is not caused byvascular defects, but is due to defective VEGF-mediated survival signalsto motor neurons. The present invention relates in particular to theisoform VEGF₁₆₅ which stimulates survival of motor neurons via bindingto neuropilin-1, a receptor known to bind semaphorin-3A which isimplicated in axon retraction and neuronal death, and the VEGFReceptor-2. The present invention thus relates to the usage of VEGF, inparticular VEGF₁₆₅, for the treatment of neuron disorders and relates,in addition, to the usage of polymorphisms in the VEGF promotor fordiagnosing the latter disorders.

BACKGROUND OF THE INVENTION

[0002] VEGF is a key player in the formation of new blood vessels(angiogenesis) during embryonic development as well as in a variety ofpathological conditions^(1,2). Although VEGF primarily stimulatesendothelial cells, it may also act on other cell types. Indeed, VEGF,VEGF receptor-1 (VEGFR-1/Flt1) and VEGF receptor-2 (VEGFR-2/KDR/Flk1)have recently been implicated in stroke^(3,4), spinal cord ischemia⁵,and in ischemic and diabetic neuropathy⁶, WO 0062798. However, thelatter molecules act predominantly via affecting vascular growth orfunction and a direct effect of VEGF on for example neuronal cells hasnot been shown^(11,12). Moreover, the in vivo relevance of such a directeffect is not validated.

[0003] Ischemia plays an essential role in the pathogenesis ofneurological disorders, acutely during stroke and chronically duringaging and several neurodegenerative disorders such as Alzheimer'sdisease, Parkinson's disease and Huntington disease. Neurons areparticularly vulnerable to oxidative stress by free radicals (generatedduring ischemia/reperfusion) because of their high oxygen consumptionrate, abundant lipid content, and relative paucity of antioxidantenzymes compared to other organs¹⁶.

[0004] Cumulative oxidative damage due to a toxic gain of function ofmutant Cu, Zn-superoxide dismutase (SODI) participates in degenerationof motor neurons in a number of patients with familial amyotrophiclateral sclerosis (ALS)^(17,18). ALS affects 5 to 10 individuals per100,000 people worldwide during the second half of their life, isprogressive, usually fatal within 5 years after onset of symptoms, anduntreatable¹⁷⁻¹⁹. Ninety to 95% of cases are sporadic. Although themechanisms underlying sporadic ALS remain unknown, evidence suggeststhat oxidative injury, similar to that caused by SOD1 mutations, plays apathogenetic role^(18,20,21).

[0005] In response to hypoxia, ‘survival’ responses are initiated,including the production of stress hormones, erythropoietin, glycolyticenzymes and angiogenic molecules such as VEGF^(22,23). Hypoxia-induciblefactors (HIFs) play an essential role in mediating this feedbackresponse via binding to a defined hypoxia-response element (HRE) in thepromotor of these genes²³. Hypoxia is a predominant regulator of VEGFexpression as induction of VEGF expression is rapid (minutes),significant (>10-fold) and responsive to minimal changes inoxygen^(22,23). Surprisingly, little attention has been paid to thepossible role of hypoxia and HIFs in the initiation of feedback survivalmechanisms in the nervous system. While several neurotrophic moleculeshave been identified^(24,25), few have been shown to be regulated byhypoxia. In this regard, it remains unknown whether hypoxic regulationof VEGF provides neuroprotection, independently of its angiogenicactivity.

[0006] Further in the nervous system, motor neurons are a well-defined,although heterogeneous group of cells responsible for transmittinginformation from the central nervous system to the locomotor system.Spinal motor neurons are specified by soluble factors produced bystructures adjacent to the primordial spinal cord, signalling throughhomeodomain proteins. Axonal pathfinding is regulated by cell-surfacereceptors that interact with extracellular ligands and once synapticconnections have formed, the survival of the somatic motor neuron isdependent on the provision of target-derived growth factors, althoughnon-target-derived factors, produced by either astrocytes or Schwanncells, are also potentially implicated. Somatic motor neurondegeneration leads to profound disability, and multiple pathogeneticmechanisms including aberrant growth factor signalling, abnormalneurofilament accumulation, excitotoxicity; autoimmunity have beenpostulated to be responsible. Even when specific deficits have beenidentified, for example, mutations of the superoxide dismutase-1 gene infamilial amyotrophic lateral sclerosis and polyglutamine expansion ofthe androgen receptor in spinal and bulbar muscular atrophy, themechanisms by which somatic mortor neuronal degeneration occurs remainunclear. In order to treat motor system degeneration effectively, weneed to understand these mechanisms more thoroughly. Although it hasbeen shown in the art that VEGF has neurotrophic actions on culturedmouse superior cervical ganglia and on dorsal root ganglia (Sondell M.et al. Journal of Neuroscience, (1999) 19, 5731), no studies areavailable about the possible role of VEGF on motor neurons. The presentinvention demonstrates that VEGF has a trophic role for neurons, inparticular motor neurons, and unveils that defective hypoxic regulationof VEGF predisposes to neuron degeneration. Moreover, the presentinvention indicates that VEGF is a therapeutic agent for the treatmentof motor neuron disorders and relates to the usage of polymorphisms inthe VEGF promotor for diagnosing neuron disorders.

AIMS OF THE INVENTION

[0007] The present invention aims at providing research tools,diagnostics and therapeutics in order to improve the health andwell-being of patients suffering from neural disorders. In particular,the present invention aims at providing the usage of VEGF, or homologuesor fragments thereof, in order to treat patients suffering fromAlzheimer disease, Parkinson's disease, Huntington disease, chronicischemic brain disease, amyotrophic lateral sclerosis, amyotrophiclateral sclerosis-like diseases and other degenerative neuron, inparticular motor neuron, disorders. More particularly, the presentinvention aims at providing the usage of VEGF₁₆₅ to prevent death ofmotor neurons in the spinal cord. The present invention also aims atproviding receptors, such as neuropilin-1 and the vascular endothelialgrowth factor receptor-2 (VEGFR-2), which specifically bind to VEGF andwhich can be used to screen for other molecules binding to it. In otherwords, the present invention aims at providing therapeutics whichstimulate survival of neurons or which inhibit death of neurons inducedby, for example, semaphorin 3A. The present invention further aims atproviding an animal which is characterized by having an altered (i.e.impaired or non-functional) hypoxia-induced VEGF expression compared toit's wild-type counterpart and which can be used as a research tool toscreen for therapeutics as mentioned above. The present inventionfinally aims at providing polymorfisms in the VEGF promoter region, suchas in the Hypoxia Responsive Element, which can be used to identifyindividuals prone to develop a neuron disorder or to treat neurondisorder patients via gene therapy.

FIGURE LEGENDS

[0008]FIG. 1: Targeting of the VEGF gene and muscle weakness inVEGF^(m/m) mice.

[0009] Strategy to delete the HIF-1a binding-element in the VEGFpromoter. The targeting vector pBSK.VEGF^(m), the wild type (VEGF^(WT))VEGF allele, the homologously recombined (VEGF^(neo)) VEGF allele, andthe modified VEGF^(m) allele after Cre-excision of the floxed neocassette are shown. Probes are indicated by solid bars. HRE:hypoxia-response element to which HIF-1alfa binds; the asterisk and “m”denote the HRE deletion.

[0010]FIG. 2: Neurotrophic role of VEGF.

[0011] A, VEGF₁₆₅, but not VEGF₁₂₁, protects SCN34 motor neurons againstapoptosis (quantified by oligonucleosomes) induced by TNF-alfa (50ng/ml). The survival activity of VEGF₁₆₅ is comparable to that of bFGFor TGF-β1. B, VEGF₁₆₅ also protects SCN34 cells against apoptosisinduced by hypoxia, H₂O₂, or serum deprivation. *:p<0.05 versus 0.01ng/ml VEGF. C, The survival effect of VEGF₁₆₅ (100 ng/ml) is blocked byantibodies (Ab; 50 μg/ml) against VEGFR-2 (R2) and neuropilin-1 (NP1),but not to VEGFR-1 (R1), neuropilin-2 (NP2), or control (ctr) IgG's.Apoptosis was induced by serum starvation (0.5%). None of the antibodiesmodified the baseline level of apoptosis in the absence of VEGF.*:p<0.05 versus control IgG.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention shows that deletion of the hypoxia-responseelement in the VEGF promotor effectively abrogates hypoxic induction ofVEGF. Based on the well-known role of VEGF in angiogenesis, it wasanticipated that VEGF^(m/m) mice would suffer impaired VEGF-mediatedangiogenesis. Vascular defects do indeed appear to contribute to thelethality of VEGF^(m/m) embryos but, surprisingly, there are no signs ofvascular insufficiency in surviving VEGF^(m/m) mice under baselineconditions.

[0013] Furthermore, the neuropathy which is seen in adult VEGF^(m/m)mice is also not due to vascular insufficiency because of the followingfindings: (i) the number, differentiation and ultrastructure ofendothelial cells in the spinal cord, peripheral nerves and muscles ofthose mice are normal; (ii) endoneural perfusion is normal without signsof leakiness; (iii) pimonidazole staining of spinal cords after hypoxiais comparable with wt-mice; (iv) infarcts or microangiopathy, typicallyfound in diabetic patients with ischemic neuropathy⁴⁷, are absent; (v)axonal lesions are not only present at the center (prone to ischemia),but also at the periphery of the nerves; (vi) degenerating motor neuronslay frequently in the immediate vicinity of normal capillaries; and(vii) other causes of ischemia including cardiac failure, anemia, orpulmonary insufficiency are excluded.

[0014] The present invention thus relates to a novel transgenic mousemodel with an impaired hypoxic upregulation of VEGF and characterized byhaving a predisposition to adult onset progressive motor neurondegeneration with neuropathological features, reminiscent of amyotrophiclateral sclerosis. In this novel mouse model the neuropathy is notcaused by vascular defects, but by deprivation of motor neurons from theneurotrophic effect of VEGF. It should be clear however that the presentinvention not only relates to a novel transgenic mouse, but alsoencompasses any non-human transgenic animal (such as a rat, dog, rabbit,non-human primate, etc.) which is characterized by having an impaired ornon-functional hypoxia-induced VEGF expression compared to theirwild-type counterparts. The present invention has significant medicalimplications. First, the genetic etiology of degenerative motor neurondisorders remains undetermined. In less than 2% of ALS cases, mutationsin the SODI gene underlie the disease, but the pathogenesis of theremaining 98% remains unknown. Our findings indicate that abnormal generegulation—not function—of VEGF constitutes a novel risk factor formotor neuron degeneration, and compels a search for genetic alterationsthat affect VEGF gene regulation. Even in ALS patients with a SOD1mutation, genetically determined differences in VEGF gene regulation mayexplain the significant intrafamilial phenotypic variability. Second,there is no medical treatment for ALS to date. Our data demonstrate thatVEGF has therapeutic value for motor neuron disorders. The availabilityof an animal model with characteristics of familial ALS (transgenicexpression of mutant SOD1) provides an essential research tool. Ourfindings also indicate that VEGF₁₆₅ protect cortical neurons againstN-methyl-D-aspartate. Third, the present VEGF^(m/m) mouse model of adultonset motor neuron degeneration reflects several clinical andneuropathological features of ALS (progressive muscle atrophy due todegeneration of spinal motor neurons, characterized by neurofilamentinclusions in the perikaryon and axonal swellings^(17-19,32-34)). TheVEGF^(m/m) mouse is therefore a suitable model for evaluation oftherapeutic strategies. Fourth, our data militate for caution againstlong-term use of VEGF-antagonists (currently being tested for treatmentof cancer, diabetes, and rheumatoid arthritis), as they can predisposeto motor neuron degeneration.

[0015] The present invention also indicates that VEGF, or homologues,derivates or fragments thereof, can be used to manufacture a medicamentfor the treatment of neuron disorders, and specifically for thetreatment of neuronopathies and more specifically for the treatment ofmotor neuron disorders and even more specifically for the treatment ofamyotrophic lateral sclerosis and amyotrophic lateral sclerosis-likediseases. In another embodiment VEGF, or homologues, derivates orfragments thereof, can be used to manufacture a medicament to preventthe death of motor neurons in the spinal cord. In a particularembodiment the VEGF₁₆₅-isoform can be used for the treatment of motorneuron disorders.

[0016] By ‘neuron disorders’ it is meant any physiological dysfunctionor death of neurons present in the central nervous system. A non-limitedlist of such disorders comprises dementia, frontotemporal lobe dementia,Alzheimer's disease, Parkinson's disease, Huntington's disease, priondiseases, neuronopathies and motor neuron disorders. ‘Neuronopathies’are characterized by neuronal cell death of motor neurons or sensoryneurons and hence neuronopathies can be subdivided in motor and sensoryneuron disorders. Motor Neuron Disease (MND) or motor neuron disordersis a group of diseases (disorders) involving the degeneration of theanterior horn cells, nerves in the central nervous system that controlmuscle activity. This leads to gradual weakening and eventually wastingof the musculature (atrophy). Diseases of the motor neuron areclassified according to upper motor neuron (UMN) and/or lower motorneuron (LMN) involvement. Upper motor neurons originate in the brain, inparticular, the motor cortex, and they synapse either directly orindirectly onto lower motor neurons. Upper motor neurons are moreaccurately referred to as pre-motor neurons, and they are responsiblefor conveying descending commands for movement. Lower motor neurons aredivisable into two catergories: visceral and somatic motor neurons.Visceral motor neurons are autonomic pre-ganglionic neurons thatregulate the activity of ganglionic neurons, which innervate glands,blood vessels, and smooth muscle. Somatic motor neurons innervateskeletal muscle and include first, anterior horn cells, which as thename implies, are located in the anterior horn of the spinal cord, andsecond, lower motor neurons located in the cranial nerve nuclei.Amyotrophic lateral sclerosis or ALS is the most frequent form(accounting for around 80% of all cases) of motor neuron disoreders. ALSis known as Lou Gehrig's disease, named after the famous Yankee baseballplayer. The initial symptoms of ALS are weakness in the hands and legsand often fasciculation of the affected muscles. Whichever limbs areaffected first, all four limbs are affected eventually. Damage to theupper motor neurons produces muscle weakness, spasticity and hyperactivedeep tendon reflexes. Lower motor neuron damage produces muscle weaknesswith atrophy, fasciculations, flaccidity and decreased deep tendonreflexes. ALS has features of both upper and lower motor neurons of thecranial nerves, therefore symptoms are isolated to the head and neck.Some patients will also display UMN involvement of the cranial nervesand if this is the sole manifestation it is referred to as Pseudobulbarpulsy. Spinal muscular atrophy or progressive muscular atrophy is a MNDthat does not involve the cranial nerves and is due to lower motorneuron degeneration. Shy-Drager syndrome is characterized by posturalhypotension, incontinence, sweating, muscle rigidity and tremor, and bythe loss of neurones from the thoracic nuclei in the spinal cord fromwhich sympathetic fibres originate. Destructive lesions of the spinalcord result in the loss of anterior horn cells. This is seen inmyelomeningocele and in syringomyelia, in which a large fluid-filledcyst forms in the centre of the cervical spinal cord. Poliomyelitisvirus infection also destroys anterior horn cells. Spinal cord tumoursmay locally damage anterior horn cells either by growth within the cord(gliomas) or by compression of the spinal cord from the outside(meningiomas, schwannomas, metastatic carcinoma, lymphomas).

[0017] Dorsal root ganglion cells may be damaged by herpex simplex andvaricella-zoster viruses. Such infections are associated with avesicular rash in the skin regions supplied by those neurones. A similarloss of sensory neurones is observed in ataxia telangiectasia, adisorder associated with progressive cerebellar ataxia and symmetricaltelangiectases of the skin and conjunctiva. Neuronal loss from autonomicganglia is observed in amyloid neuropathies and in diabetes.

[0018] A normal number of capillaries developed in VEGF^(m/m) skeletalmuscle, but their lumen size was reduced. Irrespective of whether thesmaller capillaries were the cause or consequence of the reduced musclegrowth, oxygenation was normal and there were no signs of ischemia inVEGF^(m/m) muscle, indicating that perfusion matched the metabolicdemands of the muscle fibers. VEGF is able to induce vasodilation whichcould result in structural vascular remodeling (Laitinen M. et al.(1997) Hum Gene Ther 8, 1737) but VEGF levels in normoxic and hypoxicVEGF^(m/m) muscle were normal. The normal VEGF and reduced IGF-1 levelsmay suggest that growth of muscle fibers and not of vessels wasprimarily affected. In contrast, neuronal perfusion was reduced by 50%in VEGF^(m/m) mice, despite a normal number, size and differentiation ofthe capillaries, and a normal hypercapnic vasoreactive response. Whyperfusion is reduced in some but not in other organs in VEGF^(m/m) miceand whether these organ-specific perfusion deficits relate to thevariably reduced baseline and hypoxic VEGF levels in these organs remainto be determined. In contrast to skeletal muscle where the vasculatureexpands almost 10-fold, the neuronal vascular network expands less butprimarily remodels after birth (Feher G. et al. (1996) Brain Res DevBrain Res 91, 209). VEGF has been implicated in the remodeling of theprimitive (poorly perfused) capillary plexus at birth to a functionallyperfused vasculature in the adult (Ogunshola et al. (2000) Brain Res DevBrain Res 119, 139). An intriguing question is therefore whether thereduced neuronal VEGF levels in VEGF^(m/m) mice reduced neuronalperfusion via impaired vascular remodeling. Irrespective of themechanism, the neuronal hypoperfusion in VEGF^(m/m) mice might havecontributed to the stunted growth and infertility, for instance byimpairing secretion of hypothalamic factors. Mice with hypothalamic orpituitary defects are smaller and sterile (Chandrashekar V. et al.(1996) Biol Reprod 54, 1002). The reduced IGF-1 plasma levels areconsistent with such hypothesis.

[0019] While a reduction of neuronal perfusion by 50% did not predisposeVEGF^(m/m) mice to neuronal infarcts, it likely caused chronic neuronalischemia. Animal models of chronic spinal cord ischemia are notavailable, but acute spinal cord ischemia causes significant motorneuron degeneration (Lang-Lazdunski, L. et al. (2000) Stroke 31, 208).Surgically induced cerebral perfusion deficits caused cognitive defectsbut spared rats from motoric dysfunction, and variably caused histologicsigns of neuronal loss (Ohta H. et al (1997) Neuroscience 79, 1039). Ananimal model of spontaneous chronic neuronal ischemia is, however, notavailable. Thus, in a specific embodiment the invention provides a modelfor chronic spinal cord ischemia.

[0020] The VEGF^(m/m) mouse model promises to be fruitful for studyingthe consequences of neuro-vascular insufficiency on cognitive functionand on the progression of neurodegenerative disorders. In a specificembodiment the invention provides a model for cognitive dysfunction andin another specific embodiment the VEGF^(m/m) mouse model is useful tobreed with current mouse models known in the art for neurodegenerativedisorders, for example models for Alzheimers Disease (Bornemann et al.(2000) Ann NY Acad Sci 908, 260, Van Leuven F. (2000) Prog Neurobiol 61,305, Sommer B. et al. (2000) Rev Neurosci 11, 47).

[0021] A diminished nervous blood flow in the brain can lead to brainischemia. Brain ischemia is a process of delayed neuronal cell death andnot an instantaneous event. A diminished cerebral blood flow initiates aseries of events (the “ischemic cascade”) that can lead to celldestruction. The goal of neuroprotection is to intervene in the processthat ischemic neurons undergo as part of the final common pathway ofcell death. The ischemic cascade has been intensively studied, andalthough it has not been completely delineated, certain reproducibleaspects are recognized. The normal amount of perfusion to human braingray matter is 60 to 70 mL/100 g of brain tissue/min. When perfusiondecreases to <25 mL/100 g/min, the neuron is no longer able to maintainaerobic respiration. The mitochondria are forced to switch over toanaerobic respiration, and large amounts of lactic acid are generated.This metabolic by-product accumulates in the extracellular regions andcauses a local change in the pH level. This fundamental change in theenvironment surrounding ischemic cells has been confirmed in humans bymagnetic resonance spectroscopy and by single photon emission computedtomography (SPECT). Many studies have focussed on stroke as a model forbrain ischemia. However, recently chronic reductions in cerebral bloodflow have been observed to be associated with aging and progressiveneurodegenerative disorders which can precipitate cognitive failure(Bennet et al. (1998) Neuroreport 9, 161). For example regional cerebralblood flow abnormalities to the frontal and temporal regions areobserved in depressed patients with cognitive impairment (Dolan et al.(1992) J Neurol Neurosurg Psychiatry 9, 768, Ritchie et al. (1999) AgeAgeing 28, 385). In Alzheimer's disease (AD), an example of aneurodegenerative disorder, an impaired cerebral perfusion originates inthe microvasculature which affects the optimal delivery of glucose andoxygen and results in a breakdown of metabolic pathways in brain cellssuch as in the biosynthetic and synaptic pathways. It is proposed thattwo factors need to be present before cognitive dysfunction andneurodegeneration is expressed in AD brain, advanced aging, and thepresence of a specific condition that further lowers cerebral perfusion(de la Torre (1999) Acta Neuropathol 98, 1). Further in AD a criticalthreshold cerebral hypoperfusion is a self-perpetuating, contained andprogressive circulatory insufficiency that will destabilize neurons,synapses, neurotransmission and cognitive function, creating in its wakea neurodegenerative process characterized by the formation of senileplaques, neurofibrillary tangles and amyloid angiopathy.

[0022] Cognition is referred to the process involved in knowing, or theact of knowing, which in its completeness includes perception andjudgement. Cognition includes every mental process that can be describedas an experience of knowing as distinguished from an experience offeeling or of willing. It includes, in short, all processes ofconsciousness by which knowledge is built up, including perceiving,recognizing, conceiving, and reasoning. The essence of cognition isjudgement, in which a certain object is distinguished from other objectsand is characterized by some concept or concepts. Cognitive disorders orcognitive dysfunction are disturbances in the mental process related tocognition. An overview of cognitive disorders (also called amnesticdisorders) can be found in the Diagnostic and Statistical Manual ofMental Disorders (DSM-IV™) (ISBN 0890420629).

[0023] In a specific embodiment the novel VEGF^(m/m) mouse model can beused to identify and/or to test molecules to prevent and/or to treatneuronal ischemia or neurodegenerative disorders and/or cognitivedysfuntion.

[0024] In another embodiment the present invention further indicatesthat VEGF, or homologues, derivatives or fragments thereof, can be usedfor the manufacture or a medicament to prevent and/or to treat neuronalischemia such as brain ischemia. And in yet another embodiment VEGF, orhomologues, derivatives or fragments thereof, can be used for themanufacture or a medicament to prevent and/or to treat cognitivedysfunction.

[0025] VEGF and homologues such as VEGF-B, VEGF-C, VEGF-D and PLGF aredescribed in detail in Neufeld G. et al, Faseb Journal, 13, 9-22, 1999,Korpelainen E. I. et al, Curr. Opin. Cell. Biol. 10, 159-164, 1998 andin Joukov, V. et al. J. Cell. Physiol. 173, 211-215, 1997. Inparticular, certain of the VEGF genes, homologues, fragments, andderivatives thereof that are useful for practicing the claimed inventionare described in GenBank Accession Nos. NM 003376 (“Homo sapiensvascular endothelial growth factor (VEGF) mRNA”); NM 003377 (“Homosapiens vascular endothelial growth factor B (VEGFB) mRNA”); NM 005429(“Homo sapiens vascular endothelial growth factor C (VEGFC) mRNA”); NM004469 (“Homo sapiens c-fos induced growth factor (vascular endothelialgrowth factor D) (FIGF) mRNA); AF 024710 (“Homo sapiens vascular growthfactor (VEGF₁₆₅)) mRNA, 3′UTR, mRNA sequence”); and U.S. Pat. No.6,013,780 (“VEGF₁₄₅ expression vectors”); U.S. Pat. No. 5,935,820(“Polynucleotides encoding vascular endothelial growth factor 2”); U.S.Pat. No. 5,607,918 (“vascular endothelial growth factor-B and DNA codingtherefore”). The preferred nucleic acids of the invention encode theabove-mentioned angiogenic growth factor polypeptides, as well as theirhomologues and alleles and functionally equivalent fragments or variantsof the foregoing. For example, human VEGF 1 (VEGF A) exists in fourprincipal isoforms, phVEGF₁₂₁; phVEGF₁₄₅; phVEGF₁₆₅; and phVEGF₁₈₉.Preferably, the VEGF nucleic acid has the nucleotide sequence encodingan intact human angiogenic growth factor polypeptide, i.e., the completecoding sequence of the gene encoding a human VEGF; however the inventionalso embraces the use of nucleic acids encoding fragments of an intactVEGF.

[0026] Homologues and alleles of the nucleic acid and amino acidsequences reported for the VEGF genes, such as those mentioned herein,also are also within the scope of the present invention. In addition,nucleic acids of the invention include nucleic acids which code for theVEGF polypeptides having the sequences reported in the public databasesand/or literature, but which differ from the naturally occurring nucleicacid sequences in codon sequence due to the degeneracy of the geneticcode. The invention also embraces isolated functionally equivalentfragments, variants, and analogs of the foregoing nucleic acids;proteins and peptides coded for by any of the foregoing nucleic acids;and complements of the foregoing nucleic acids. ‘Functionally’ meansthat the fragments, variants and analogs must have the capacity to treata neuron disorder and in particular a motor neuron disorder.

[0027] The term ‘derivatives’ refers to any variant, mutant or peptidecomposition of VEGF, which retains the capacity, or can be used, totreat degenerative motor neuron disorders as defined above. The latterterm also includes post-translational modifications of the amino acidsequences of VEGF such as glycosylation, acetylation, phosphorylation,modifications with fatty acids and the like. Included within thedefinition are, for example, amino acid sequences containing one or moreanalogues of an amino acid (including unnatural amino acids), amino acidsequences with substituted linkages, peptides containing disulfide bondsbetween cysteine residues, biotinylated amino acid sequences as well asother modifications known in the art. The term thus includes any proteinor peptide having an amino acid residue sequence substantially identicalto a sequence specifically shown herein in which one or more residueshave been conservatively substituted with a biologically equivalentresidue. Examples of conservative substitutions include the substitutionof one-polar (hydrophobic) residue such as isoleucine, valine, leucineor methionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagines, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another. The phrase “conservative substitution” alsoincludes the use of a chemically derivatized residue in place of anon-derivatized residue provided that the resulting protein or peptideis biologically equivalent to the protein or peptide of the invention.

[0028] ‘Chemical derivative’ refers to a protein or peptide having oneor more residues chemically derivatized by reaction of a functional sidegroup. Examples of such derivatized molecules, include but are notlimited to, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides” p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloracetyl groups orformyl groups. Free carboxyl groups may be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-imbenzylhistidine. Also included as chemical derivatives arethose proteins or peptides, which contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For example: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. The proteins or peptides of thepresent invention also include any protein or peptide having one or moreadditions and/or deletions or residues relative to the sequence of apeptide whose sequence is shown herein, so long as the peptide isbiologically equivalent to the proteins or peptides of the invention.When percentage of sequence identity is used in reference topolypeptides (i.e. homologues), it is recognized that residue positionswhich are not identical often differ by conservative aa substitutions,where aa residues are substituted for other aa residues with similarchemical properties (for example charge or hydrophobicity) and thereforedo not change the functional properties of the polypeptide. Wheresequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Means for making this adjustment are well known tothose skilled in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example (and asdescribed in WO 97/31116 to Rybak et al.), where an identical aa isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. In this regard, it should be clear that polypeptides, or partsthereof, comprising an aa sequence with at least 55%, preferably 75%,more preferably 85% or most preferably 90% sequence identity with theamino acid sequence of VEGF, or parts thereof, fall within the scope ofthe present invention. It should also be clear that polypepuides whichare immunologically reactive with antibodies raised against VEGF, orparts thereof, fall within the scope of the present invention.

[0029] The term ‘fragments of VEGF’ refers to any fragment, includingany modification of said fragment as described above, which retains thecapacity, or can be used, to treat neuron disorders and in particularmotor neuron disorders.

[0030] The terms ‘pharmaceutical composition’ or ‘medicament’ or ‘usefor the manufacture of a medicament to treat’ relate to a compositioncomprising VEGF or homologues, derivatives or fragments thereof asdescribed above and a pharmaceutically acceptable carrier or excipient(both terms can be used interchangeably) to treat diseases as indicatedabove. Suitable carriers or excipients known to the skilled man aresaline, Ringer's solution, dextrose solution, Hank's solution, fixedoils, ethyl oleate, 5% dextrose in saline, substances that enhanceisotonicity and chemical stability, buffers and preservatives. Othersuitable carriers include any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids and amino acid copolymers. The‘medicament’ may be administered by any suitable method within theknowledge of the skilled man. The preferred route of administration isparenterally. In parental administration, the medicament of thisinvention will be formulated in a unit dosage injectable form such as asolution, suspension or emulsion, in association with thepharmaceutically acceptable excipients as defined above. However, thedosage and mode of administration will depend on the individual.Generally, the medicament is administered so that the protein,polypeptide, peptide of the present invention is given at a dose between1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, mostpreferably between 0.1 and 2 mg/kg. Preferably, it is given as a bolusdose. Continuous infusion may also be used and includes continuoussubcutaneous delivery via an osmotic minipump. If so, the medicament maybe infused at a dose between 5 and 20 μg/kg/minute, more preferablybetween 7 and 15 μg/kg/minute.

[0031] In a particularly preferred embodiment the infusion with acomposition comprising VEGF or homologues, derivatives or fragmentsthereof is intrathecal. Intrathecal administration can for example beperformed by means of surgically implanting a pump and running acatheter to the spine. It should be mentioned that intrathecaladministration of VEGF or homologues, derivatives or fragments thereofis a particularly important aspect of the present invention. Indeed,since we have shown that VEGF has a neurotrophic aspect on neurons andmore particularly on motor neurons, intrathecal administration is apreferred way. This is in contrast with WO 0062798 were therapeuticangiogenesis is aimed at in order to treat ischemic peripheralneuropathy.

[0032] Instead of delivering VEGF or a homologue, derivative or fragmentthereof, as a protein to a patient in need for treatment of a neurondisorder or more particularly a motor neuron disorder, also a nucleicacid encoding VEGF, or a homologue, derivative or fragment thereof canbe delivered to said patient. In that case the nucleic acid encodingVEGF or homologue, derivative or fragment thereof, can be operativelycoupled to a promoter that can express said angiogenic growth factor ina target cell (e.g., an endothelial cell, a nerve cell, a muscle cell, aneuron, a motor neuron). Often the nucleic acid is contained in anappropriate expression vector (e.g., plasmid, adenoviral vector,modified adenoviral vector, retroviral vector, liposome) to moreefficiently genetically modify the target cell and achieve expression ofsaid angiogenic growth factor. For example, in WO 9831395 the selectivetransfer of genes into motor neurons is fully described.

[0033] In another embodiment of the invention it is shown that theVEGF₁₆₅ isoform, but not the VEGF₁₂₁ isoform, provides neuroprotectionvia binding to neuropilin-1 and VEGFR-2.

[0034] In yet another embodiment of the invention inhibitors of Sema3A,a molecule which is implicated in neuronal apoptosis⁴³ and axonretraction⁴⁴, and inhibits binding of VEGF₁₆₅ to neuropilin-1⁹, can bemade and used for the treatment of neuron disorders. Neuropilin-1 alsobinds Sema3A, implicated in repulsion of motor projections duringdevelopment¹¹⁻¹⁵. Neuropilin-1 and Sema3A are expressed in the ventralhorn after birth, but their role has remained enigmatic. A recent invitro study suggested a role for Sema3A in apoptosis of sympathetic andcerebellar neurons⁴³, whereas downregulation of Sema3A was suggested tobe a prerequisite for axonal regeneration after nerve injury⁴⁴.

[0035] In yet another embodiment VEGF, or homologues, derivatives orfragments thereof can be administrated for the prevention of neuronalloss or more specifically of motor neuronal loss in the spinal cord infor example surgical indications where an ischemic insult to neurons ormotor neurons can be expected. The initiation of neuroprotectivepathways during hypoxia is required, as these vital post-mitotic motorneurons cannot regenerate after a lethal hypoxic insult. In this regardonly a few neuroprotective molecules such as NGF, bFGF, TGFβ1⁵²⁻⁵⁴ havebeen characterized. The present invention clearly indicates that VEGF isa potent neuroprotective agent, as regulation of its expression byhypoxia is rapid (minutes), significant (>10-fold) and sensitive tosmall changes in oxygen. The absence of neuroprotective VEGF responsesin VEGF^(m/m) mice—even though they might only occur transiently, butrepetetively—may explain why motor neurons in these mice ultimatelydegenerate after cumulative sublethal mini-insults of hypoxia.

[0036] Neuropilin-1 (NP-1), a receptor for the VEGF₁₆₅ isoform^(9,10)and for the neurorepellant semaphorin 3A (Sema3A)¹¹⁻¹³ is shown to beessential for guiding neuronal projections during embryonicpatterning¹¹⁻¹⁵. However, it is not known if NP-1 and/or Sema3A have anyrole in neuronal function after birth. In a further embodiment theinvention further provides methods for identifying compounds ormolecules which bind on the neuropilin receptor and VEGFR-2 andstimulate the survival of neurons and more particularly motor neurons.In this invention the results show that VEGF₁₆₅, via binding toneuropilin-1 and VEGFR-2, mediates survival of NSC34 motor neurons. Bothreceptors are expressed on motor neurons in adult spinal cords in vivo,and are therefore accessible to VEGF₁₆₅, produced by the motor neuronitself or by other nearby cells. Neuropilin-1 and VEGFR-2 act asco-receptors in stimulating endothelial cell motility^(9,10) and alsocooperate in mediating neuronal survival. These methods are alsoreferred to as ‘drug screening assays’ or ‘bioassays’ and typicallyinclude the step of screening a candidate/test compound or agent for theability to interact with (e.g. bind to) neuropilin-1 and VEGFR-2.Candidate compounds or agents, which have this ability, can be used asdrugs to treat degenerative disorders. Candidate/test compounds such assmall molecules, e.g. small organic molecules, and other drug candidatescan be obtained, for example, from combinatorial and natural productlibraries.

[0037] The invention also provides methods for identifying compounds oragents which can be used to treat degenerative neurons. These methodsare also referred to as ‘drug screening assays’ or ‘bioassays’ andtypically include the step of screening a candidate/test compound oragent for the ability to interact with (e.g., bind to) neuropilin-1 andVEGFR-2. Candidate/test compounds or agents which have this ability, canbe used as drugs to treat degenerative neuron disorders. Candidate/testcompounds such as small molecules, e.g., small organic molecules, andother drug candidates can be obtained, for example, from combinatorialand natural product libraries. In one embodiment, the invention providesassays for screening candidate/test compounds which interact with (e.g.,bind to) neuropilin-1 and VEGFR-2. Typically, the assays are cell-freeassays which include the steps of combining neuropilin-1 and VEGFR-2 anda candidate/test compound, e.g., under conditions which allow forinteraction of (e.g. binding on the candidate/test compound withneuropilin-1 and VEGFR-2 to form a complex, and detecting the formationof a complex, in which the ability of the candidate compound to interactwith neuropilin-1 and VEGFR-2 is indicated by the presence of thecandidate compound in the complex. Formation of complexes between theneuropilin-1 and the candidate compound can be quantitated, for example,using standard immunoassays. The neuropilin-1 employed in such a testmay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. In another embodiment, theinvention provides screening assays to identify candidate/test compoundswhich stimulate neuropilin-1 and VEGFR-2 or inhibit binding of sema3A toneuropilin-1 and/or VEGFR-2. Typically, the assays are cell-free assayswhich include the steps of combining neuropilin-1 and VEGFR-2 of thepresent invention or fragments thereof, and a candidate/test compound,e.g., under conditions wherein but for the presence of the candidatecompound, the neuropilin-1 and, VEGFR-2 or a biologically active portionthereof interacts with (e.g., binds to) the target molecule or theantibody, and detecting the formation of a complex which includes theneuropilin-1 and the target molecule or the antibody, or detecting theinteraction/reaction of neuropilin-1 and the target molecule orantibody. Detection of complex formation can include direct quantitationof the complex.

[0038] To perform the above described drug screening assays, it isfeasible to immobilize neuropilin-1 and VEGFR-2 or its (their) targetmolecule(s) to facilitate separation of complexes from uncomplexed formsof one or both of the proteins, as well as to accommodate automations ofthe assay. Interaction (e.g., binding of of neuropilin-1 and VEGFR-2 toa target molecule can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and microcentrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows theprotein to be bound to a matrix. For example, neuropilin-1-His taggedcan be adsorbed onto Ni-NTA microtitre plates (Paborsky et al., 1996),or neuropilin-1-ProtA fusions adsorbed to IgG, which are then combinedwith the cell lysates (e.g., ³⁵S-labeled) and the candidate compound,and the mixture incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the plates are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of neuropilin-1 binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques.Other techniques for immobilizing protein on matrices can also be usedin the drug screening assays of the invention. For example, eitherneuropilin-1 and VEGFR-2 or its target molecules can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylatedneuropilin-1 and VEGFR-2 can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with neuropilin-1 but which do notinterfere with binding of the protein to its target molecule can bederivatized to the wells of the plate, and neuropilin-1 and VEGFR-2trapped in the wells by antibody conjugation. As described above,preparations of a neuropilin-1-binding protein and a candidate compoundare incubated in the neuropilin-1-presenting wells of the plate, and theamount of complex trapped in the well can be quantitated. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the neuropilin-1 target molecule andVEGFR-2-target molecule, or which are reactive with neuropilin-1 andVEGFR-2 and compete with the target molecule; as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule. Another technique for drug screening which provides forhigh throughput screening of compounds having suitable binding affinityto neuropilin-1 and VEGFR-2 is described in detail in. “Determination ofAmino Acid Sequence Antigenicity” by Geysen H N, WO 84103564, publishedon 13109184. In summary, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The protein test compounds are reacted withfragments of neuropilin-1 or/and VEGFR-2 and washed. Bound neuropilin-1is then detected by methods well known in the art. Purified neuropilin-1or/and VEGFR-2 can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support. This invention also contemplates theuse of competitive drug screening assays in which neutralizingantibodies capable of binding neuropilin-1 or/and VEGFR-2 specificallycompete with a test compound for binding neuropilin-1 or/and VEGFR-2. Inthis manner, the antibodies can be used to detect the presence of anyprotein, which shares one or more antigenic determinants withneuropilin-1 and VEGFR-2

[0039] No genetic mutations of the VEGF gene, resulting in genedisruption, have thus far been linked to human disease, likely becauseabsence of even a single VEGF allele is embryonically lethal^(1,2,26).Recently, however, impaired hypoxic regulation of VEGF has been shown toconstitute a risk factor for ischemic heart disease 27 Whether thisabnormal VEGF gene regulation—not function—may predispose topathological disorders is, however, not known. In another embodiment ofthe invention polymorphisms in the regulatory region of the VEGF gene,which have an influence on the hypoxic regulation of said gene, can bedetected and used diagnostically to identify patients at risk to developa neuropathy or more specifically a motor neuropathy when exposed tobrief periods of ischemia. Hypoxia-induced transcription of VEGF mRNA ismediated, at least in part, by the binding of hypoxia-inducible factor 1(HIF-1) to an HIF-1 binding site located in the VEGF promotor. By thedetection of polymorphisms that influence the hypoxic regulation of theVEGF gene it is also meant polymorphisms in the HIF-1 transcriptionfactor, additional HIF-1-like factors, upstream regulators of HIF-1 andHIF-1-like transcription factors comprising the oxygen-sensor,additional factors binding to the 5′ and 3′ untranslated region of theVEGF mRNA and in the internal ribosomal entry site present in the 5′untranslated region of VEGF (Neufeld G. et al. FASEB J. 13, 9-22, 1999).

[0040] Several procedures have been developed for scanning genes inorder to detect polymorphisms in genes. In terms of current use, many ofthe methods to scan or screen genes employ slab or capillary gelelectrophoresis for the separation and detection step in the assays.Some of these methods comprise Single strand conformational polymorphism(SSCP) (Orita et al., “Detection of Polymorphisms of Human DNA by GelElectrophoresis as Single-Stranded Conformation Polymorphisms,” Proc.Natl. Acad. Sci. USA 86, 2766 (1989)), denaturing gradient gelelectrophoresis (DGGE) (Abrams et al., “Comprehensive Detection ofSingle Base Changes in Human Genomic DNA Using Denaturing Gradient GelElectrophoresis and a GC Clamp,” Genomics 7, 463 (1990)), chemicalcleavage at mismatch (CCM) (J. A. Saleeba & R. G. H. Cotton, “ChemicalCleavage of Mismatch to Detect Mutations,” Methods in Enzymology 217,286 (1993)), enzymatic mismatch cleavage (EMC) (R. Youil et al.,“Screening for Mutations by Enzyme Mismatch Cleavage with T4Endonuclease VII,” Proc. Natl. Acad. Sci. USA 92, 87 (1995)), and“cleavase” fragment length polymorphism (CFLP). Still other methodsfocus on the use of mass spectrometry as a genetic analysis tool. Massspectrometry requires minute samples, provides extremely detailedinformation about the molecules being analyzed including high massaccuracy, and is easily automated. U.S. Pat. No. 5,965,363 describesnucleic acid analysis by means of mass spectrometric analysis.

[0041] In another embodiment of the invention the Hypoxia ResponseElement (HRE) can be used for the treatment of neuron disorders or morespecifically motor neuron disorders. VEGF, or homologous, derivates orfragments thereof, can be placed under hypoxic control by splicing saidgenes to one or more HRE elements. These constructs can then be used ingene therapy.

[0042] Gene therapy means the treatment by the delivery of therapeuticnucleic acids to patient's cells. This is extensively reviewed in Leverand Goodfellow 1995; Br. Med Bull., 51, 1-242; Culver 1995; Ledley, F.D. 1995. Hum. Gene Ther. 6, 1129. To achieve gene therapy there must bea method of delivering genes to the patient's cells and additionalmethods to ensure the effective production of any therapeutic genes.There are two general approaches to achieve gene delivery; these arenon-viral delivery and virus-mediated gene delivery. The bestcharacterized virus-mediated gene delivery system uses replicationdefective retroviruses to stably introduce genes into patients' cells.

[0043] The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and cannot be construed as to restrict the invention in anyway.

EXAMPLES

[0044] 1. Targeted Deletion of the HIF-Binding Site in the VEGF Promotor

[0045] Targeted deletion in the VEGF promotor of the hypoxia-responseelement (HRE), i.e. the binding site for the hypoxia-inducible factors(HIF) 23, was achieved using Cre/loxP-mediated targeting (FIG. 1), andconfirmed by Southern blot analysis.

[0046] Impaired hypoxic induction of VEGF in embryonic stem cells,homozygous for the HRE-deletion (VEGF^(m/m)), was confirmed by Northernblot analysis and by measurements of VEGF release during 36 h hypoxia(19±5 μg/ml after normoxia versus 45±6 μg/ml after hypoxia in VEGF^(+/+)cells, n=6, p<0.05; 11±2 μg/ml after normoxia versus 13±3 μg/ml afterhypoxia in VEGF^(m/m) cells, n=6; p=NS). Deletion of the HIF-bindingsite in the VEGF gene was as effective as deletion of the HIF-1α geneitself 28 in abolishing hypoxic upregulation of VEGF (13±4 μg/ml afternormoxia versus 14±2 μg/ml after hypoxia in HIF-1α^(−/−) cells; n=6;p=NS). VEGF^(m/m) embryos were recovered at a normal Mendelianfrequency. Of the VEGF^(m/m) mice, 30% died before birth and another 30%within the first postnatal days, while the remaining 40% survived morethan 12 months. Here, the phenotype of the surviving VEGF^(m/m) mice isdescribed; the embryonic and neonatal phenotypes will be reportedseparately.

[0047] 2. Motor Coordination and Muscular Performance in VEGF^(m/m) Mice

[0048] VEGF^(m/m) mice appeared normal until 4 months, but thereafterdeveloped symptoms of motor neuron disease. They became progressivelyless mobile, and exhibited signs of severe muscle weakness and limbparesis. Beyond six months of age, mutant mice were too weak to turnover when placed on their back, slapped their feet while walking and hada waddling gait and scoliosis. When lifted by their tails, theyreflexively contracted their limbs to the trunk and remained immobile,whereas wild type mice extended their limbs and struggled. VEGF^(m/m)mice developed a coarse fur suggestive of impaired grooming and appearedthin along their flanks and legs. Notably, when asymptomatic two monthsold VEGF^(m/m) mice were kept in a hypoxic chamber (10% O₂), theydeveloped neurological signs (difficulty in turning over, reflexcontracture when lifted by tail) within two weeks, indicating thathypoxia markedly accelerated the onset and progression of the phenotype.Beyond. 4 to 6 months of age, VEGF^(m/m) mice performed significantlyless well than wild type littermates in a number of motor coordinationand muscle performance tests²⁹, including the treadmill-wheel test, gridtest, rotating axle test and the footprint test (distance between thecentral pads of the hindfeet: 65±5 mm for VEGF^(+/+) mice versus 45±5 mmfor VEGF^(m/m) mice; n=7; p<0.05). Compared to VEGF^(+/+) mice,VEGF^(m/m) mice were significantly less active at night (number oftreadmill-wheel turns: 5400±600 for VEGF^(+/+) mice versus 2700±400 forVEGF^(m/m) mice; n=7; p<0.05) and for much shorter periods (minutes ofintense activity: 150±40 for VEGF^(+/+) mice versus 14±6 for VEGF^(m/m);n=7; p<0.05). In the ‘grid’ test (mice are placed on a grid, that issubsequently turned upside-down), five of seven VEGF^(+/+) mice hung onto the grid for at least one minute. Two VEGF^(+/+) mice moved soactively that they dropped from the rack after 23 and 45 seconds. Incontrast, four of six 20 week-old VEGF^(m/m) mice fell already off thegrid after 8 seconds, and only two mutant mice managed to hold on to thegrid by not moving at all. When testing their grip strength (mice areforced to hang with their forelimbs on a horizontal thread), allVEGF^(+/+) mice (n=7) immediately grabbed the thread with theirhindlimbs. In contrast, VEGF^(m/m) mice had difficulties in grabbing thethread with their hindlimbs, hung immobile and sagged. When they finallysucceeded (five of six mice), VEGF^(m/m) mice could not hold on to thethread and fell off. VEGF^(m/m) mice also performed worse in the‘rotating axle’ test, used to evaluate how long mice could stay on arotating axle before falling off: all but one VEGF^(+/+) mice (n=8)stayed on the axle for at least two minutes (time of analysis), whereasall of six VEGF^(m/m) mice fell off after less than a minute (53±20sec). Pain threshold, a sensory function measured as the paw-lickresponse in a hot plate test 29 was normal in VEGF^(m/m) mice (the timeto lick the front or rear paws for both genotypes was 7±1 s and 10±2 s,respectively; n=6; p=NS). However, VEGF^(+/+) mice jumped out of the boxafter 100±20 s, whereas VEGF^(m/m) mice were too weak to escape duringthis period.

[0049] 3. Muscle Atrophy in VEGF^(m/m) Mice

[0050] Skeletal muscles in VEGF^(m/m) mice beyond 4 months of age showedsigns of neurogenic atrophy. The wet weight of the plantar and dorsalflexor muscles was 170±14 mg and 92±19 mg in VEGF^(+/+) mice versus 94±5mg and 58±18 mg in VEGF^(m/m) mice (n=3; p<0.05). Initially, a variablenumber of fibers were atrophic, but in older animals, most muscle fiberswere severely atrophic, “angulated” or “elongated”, characteristics ofdenervated fibers. Muscle fiber size was decreased by more than 50% inVEGF^(m/m) mice (cross-sectional area: 1700±200 μm² in VEGF^(+/+) miceversus 700±100 μm² in VEGF^(m/m) mice; n=8; p<0.05). Regenerating musclefibers, identified by their centrally located vesicular nucleus, smallersize and desmin-immunoreactivity, were commonly observed in VEGF^(m/m)mice. Myosin ATPase staining revealed atrophy of all fiber types(type-I, -IIa, and -IIb). In contrast to the typical chessboard patternof all fiber types in VEGF^(+/+) mice, grouping of fibers of a similartype, a sign of reinnervation, was observed in VEGF^(m/m) mice. Musclespindles—involved in reflex control—were present in both genotypes(number of spindles/quadriceps section: 3.9±0.9 in VEGF^(+/+) miceversus 4.5±0.8 in VEGF^(m/m) mice; n=5; p=NS). Myopathic changes(sarcolemma desintegration, fiber necrosis, loss of muscle fibers,elevated plasma creatine kinase levels or fibrosis) were not detected inVEGF^(m/m) mice. The muscle atrophy in VEGF^(m/m) mice did not resemblethe degenerative features of primary myopathies. Indeed, there was noloss of muscle fibers and, because of shrinkage, the density of themuscle fibers was increased in VEGF^(m/m) mice (1250±190 cells/mm²) ascompared to VEGF/+mice (720±80 cells/mm²; p<0.05). Unlike in myopathies,there were no signs of fiber necrosis, sarcomere lysis or sarcolemmadisruption (ultrastructural analysis; normal titin and desmin staining;absence of intracellular albumin), fatty infiltration, fibrosis (siriusred staining), or dystrophic calcification. Plasma levels of creatinekinase (released upon myocyte death) were normal in 8 month-oldVEGF^(m/m) mice (88±20 U/ml in VEGF^(+/+) mice versus 94±9 U/ml inVEGF^(m/m) mice; n=5; p=NS). In addition, atrophy was confined toskeletal and not to cardiac muscle, and was not caused by systemicdisorders. Atrophy was confined to skeletal muscle fibers, sincecardiomyocytes were not affected (cross-sectional area: 130±10 μm² inVEGF^(+/+) mice versus 125±8 μm² in VEGF^(m/m) mice; n=5; p=NS).Structural changes in muscle fibers were also not due to infectiousdisease (pathogen-free health report), inflammatory disorders ofconnective tissue or blood vessels (no signs of vasculitis), metabolicdisorders (normal plasma glucose levels), or storage abnormalities inglycogen or lipids.

[0051] 4. Motor Neuron Degeneration in VEGF^(m/m) Mice

[0052] Evidence for a neurodegenerative process was obtained by analysisof the spinal cord and peripheral nerves. Nissl staining revealed acomparable number of neurons in the ventral horn in the spinal cord atyoung age (12 weeks) in both genotypes (neurons with a clearlyidentifiable cytoplasm/ventral horn section: 110±2 in VEGF^(+/+) miceversus 107±6 in VEGF^(m/m) mice; n=3; p=NS), indicating that deletion ofthe HIF-binding site in the VEGF promotor per se did not cause abnormalneuronal development. However, beyond 7 months of age, fewer neuronswere detected in the ventral horn in VEGF^(m/m) mice (neurons/ventralhorn section: 110±3 in VEGF^(+/+) mice versus 98±4 in VEGF^(m/m) mice;n=8; p<0.05). Immunostaining for choline acetyltransferase (ChAT), amarker of motor neurons, revealed 30% fewer motor neurons in VEGF^(m/m)than in VEGF^(+/+) mice (ChAT-positive neurons/spinal cord section: 26±2in VEGF^(+/+) mice versus 18±2 in VEGF^(m/m) mice; n=4; p=0.05). Incontrast to VEGF^(+/+) mice, the neuronal cell bodies (perikarya) andproximal axons of motor neurons in VEGF^(m/m) mice contained inclusionsof phosphorylated neurofilament (NF_(P)), a hallmark of motor neurondisease³¹ (number of NF_(P)-positive neurons/spinal cord section: nonein VEGF^(+/+) mice versus 7±2 in VEGF^(m/m) mice; n=6; p<0.05). TheseNF_(P)-positive motor neurons were smaller in size (250±20 μm²) thanNF_(P)-negative motor neurons in VEGF^(+/+) mice (500±40 μm²; n=4,p<0.05). The complexity and occurrence of dephosphorylated neurofilament(NF)-positive axons and of MAP-2-positive dendrites was comparable inboth genotypes, even though more neurons in VEGF^(m/m) mice tended toaccumulate dephosphorylated NF in their perikaryon. Focal axon swellings(‘spheroids’), also found in ALS patients^(33,34), occurred in thespinal cord in VEGF^(m/m) but not in VEGF^(+/+) mice (number of swollenaxons/spinal cord section at 31 weeks of age: none in VEGF^(+/+) miceversus 17±1 in VEGF^(m/m) mice; n=7). Swollen axons with dense axoplasmwere primarily located in the ventral horn, whereas the dorsal spinalcord or the corticospinal tracts appeared relatively spared. A fractionof these swollen axons was immunoreactive for synaptophysin, a sign ofimpaired axonal transport, and for ubiquitin, which binds damagedproteins in neurodegenerative conditions such as ALS³⁵. They oftencontained neurofilament inclusions, as revealed by Bielschowski stainingand immunostaining for phosphorylated neurofilament (NF_(P)). Comparedto VEGF^(+/+) mice, a prominent reactive astrocytosis was consistentlyobserved in the spinal cord of VEGF^(m/m) mice, but characteristicallyonly in the ventral horn. Numerous hypertrophic astrocytes accumulatedin the ventral and intermediate zones (GFAP-positive area in graymatter: 0.8±0.4% in VEGF^(+/+) mice versus 7±1% in VEGF^(m/m) mice; n=4;p<0.05), and in the ventral white matter (GFAP-positive area in whitematter: 8±3% in VEGF^(+/+) mice versus 31±2% in VEGF^(m/m) mice; n=4;p<0.05).

[0053] 5. Axon Degeneration in VEGF^(m/m) Mice

[0054] Signs of Wallerian degeneration and significant loss of largeaxons were found. Some fibers were completely replaced by the morenumerous activated macrophages, phagocytosing disrupted myelin sheets(number of F4/80-positive cells/mm²: 150±27 in VEGF^(+/+) mice versus340±20 in VEGF^(m/m) mice; n=6; p<0.05). Endoneural fibrosis andexpression of GFAP, a marker of denervated Schwann cells, were moreprominent in mutant nerves.

[0055] 6. Electrophysiology of VEGF^(m/m) Mice

[0056] Electromyographic (EMG) recordings during rest and musclecontraction revealed clear signs of denervation and reinnervation.Diffuse spontaneous activity (fibrillation potentials, isolated orsalvo's of positive sharp waves), together with polyphasic motor unitaction potentials (MUAP's) of normal amplitude, and unstable satellitepotentials were observed in the superficial gastrocnemius muscle, theparavertebral muscles and the diaphragm in VEGF^(m/m) but not inVEGF^(+/+) mice. In the diaphragm, denervation was evidenced by areduced recruitment of MUAP's during inspiration in VEGF^(m/m) mice(number of MUAP's per inspiratory burst: 124±20 in VEGF^(+/+) miceversus 56±4 in VEGF^(m/m) mice; n=7; p<0.05). Compared to the normal bi-or triphasic pattern of MUAPs in VEGF^(+/+) mice, long-durationpolyphasic MUAP's were regularly detected. The duration of inspiratorybursts in VEGF^(m/m) mice remained normal (2300±230 ms in VEGF^(+/+)mice versus 2100±300 ms in VEGF^(m/m) mice; n=7; p=NS), and theamplitude of the largest diaphragmatic MUAP's were preserved inVEGF^(m/m) mice (MUAPs with an amplitude>200 pV per inspiratory burst:14±2 in VEGF^(+/+) mice versus 11±4 in VEGF^(m/m) mice, n=7; p=NS),consistent with an ongoing process of denervation and reinnervation.Furthermore, in contrast to the innervation of individual endplates by asingle terminal axon, terminal axons in VEGF^(m/m) mice more oftenbranched as thin (presumably unmyelinated) sprouts to two or moreendplates. Latencies of compound muscle action potentials (measured fromsite of stimulation to site of recording) were somewhat increased inmutant mice (810±36 μs in VEGF^(+/+) mice versus 1030±44 μs inVEGF^(m/m) mice; n=7; p<0.05), compatible with the axonal loss. Sensorynerve function appeared electrophysiologically normal: sensory nerveaction potential (SNAP) amplitudes were 100±7 μV in VEGF^(m/m) miceversus 120±14 μV in VEGF^(m/m) mice (n=5; p=NS), and sensory nerveconduction velocities were 33±2 m/s in VEGF^(+/+) mice versus 30±2 m/sin VEGF^(m/m) mice (n=5; p=NS). These findings are consistent with apurely motor neurogenic disorder.

[0057] 7. Normal Vascular Growth in VEGF^(m/m) Mice

[0058] Because of the well-known angiogenic role of VEGF, VEGF^(m/m)mice were examined for vascular defects. However, there were no signs ofvascular insufficiency in the skeletal muscle, peripheral nerves, orspinal cord. (i) In muscle, because of the atrophy, capillary densitieswere higher in VEGF^(m/m) than in VEGF^(+/+) mice (capillaries/mm²:1200±150 in VEGF^(+/+) mice versus 1740±150 in VEGF^(m/m) mice; n=5;p<0.05). Fluoro-angiography⁶ of the diaphragm revealed comparablevascularization in both genotypes. (ii) Sciatic nerves in VEGF^(m/m)mice had a similar density of vasa nervorum (capillaries/mm²: 90±6 inVEGF^(+/+) mice versus 100±7 in VEGF^(m/m) mice; n=7; p=NS), werenormally perfused (laser doppler blood flow perfusion units: 150±15 inVEGF^(+/+) mice versus 190±12 in VEGF^(m/m) mice; n=5; p=NS), andcontained a comparable density and pattern of peri- and endoneuralvessels without signs of leakiness or obstruction (fluoro-angiography).Axonal degeneration occurred in the periphery as well as in the centerof VEGF^(m/m) nerves, arguing against ischemic neuropathy, which istypically more severe in the center³⁷. (iii) In the spinal cord,capillary densities were comparable in both genotypes in the gray matter(capillaries/mm²: 380±17 in VEGF^(+/+) mice versus 390±13 in VEGF^(m/m)mice; n=7; p=NS), and in the white matter (capillaries/mm²: 170±12 inVEGF^(+/+) mice versus 170±11 in VEGF^(m/m) mice; n=7; p=NS), withsimilar densities in ventral and dorsal horns. Endothelial cells of bothgenotypes expressed blood-brain barrier characteristics(glucose-transporter type I; Glut-1). Signs of ischemic or diabeticmicroangiopathy³⁸ were not detected in VEGF^(m/m) mice. Characteristicsigns of ischemic neuropathy (amyloid deposits, inflammatory vasculitis)or diabetic neuropathy (hyalinization of endoneural microvessels,thickening of capillary basement membrane, pericyte drop-out, lumenobstruction due to endothelial hyperplasia/hypertrophy,neovascularization, nerve infarct) were not deteced in VEGF^(m/m) mice.The muscle weakness in VEGF^(m/m) mice was not due to impairedoxygenation, nor to reduced levels of the O₂-carrier hemoglobin (normalhematological profile). In addition, echocardiographic determination ofthe circumferential fiber shortening (VCF, a measure of contractility)revealed that VEGF^(m/m) mice had normal cardiac function duringbaseline conditions (15+2 in VEGF^(+/+) mice versus 17±3 in VEGF^(m/m)mice; p=NS) and after dobutamine-stress (27±6 in VEGF^(+/+) mice versus26±6 in VEGF^(m/m) mice; p=NS). There was also no metabolic imbalance inmutant mice (normal electrolytes; plasma glucose levels: 200±10 mg/dl inVEGF^(+/+) mice versus 180±16 in VEGF^(m/m) mice, n=7; p=NS).

[0059] After exposure to hypoxia (10% O₂; 24 h), motor neurons in bothgenotypes stained comparably for the hypoxia-marker pimonidazole. Themuscle weakness in VEGF^(m/m) mice was not due to impaired oxygenation,anemia, metabolic imbalance, cardiac dysfunction, or abnormal vasculardevelopment in other organs. Collectively, there were no signs ofvascular insufficiency or ischemia in VEGF^(m/m) mice.

[0060] 8. Expression of VEGF and Neuropilin-1 in the Spinal Cord

[0061] Exposure of VEGF^(+/+) mice to hypoxia (10% O₂; 24 h) upregulatedexpression of VEGF in the spinal cord (pg/mg protein: 15±1 in normoxiaversus 94±20 in hypoxia; n=7, p<0.05), but only minimally in VEGF^(m/m)mice (pg/mg protein: 9±2 in normoxia versus 15±2 in hypoxia; n=7;p=0.06). Hypoxic induction of LDH-A (another hypoxia-inducible gene) inVEGF^(m/m) spinal cords was not abrogated (LDH-A/10³ hprt mRNA copies:230±100 during normoxia versus 1100±400 during hypoxia; n=7; p<0.05),confirming specificity of gene targeting. Similar results were obtainedin the brain. In contrast, VEGF levels were reduced in skeletal muscleafter hypoxia (pg/mg protein: 40±10 in normoxia and 22±9 in hypoxia inVEGF^(+/+) mice, n=7, p<0.05 versus 52±6 in normoxia and 23+4 in hypoxiain VEGF^(m/m) mice, n=7; p<0.05), consistent with previous observationsthat VEGF expression in response to hypoxia is tissue-specific³⁹.

[0062] 9. Neurotrophic Role of VEGF and Neuropilin-1

[0063] Apoptosis of neuronal cells has been implicated in severalneurodegenerative disorders, including ALS^(24,33). A possibleneuroprotective role of VEGF, independently of its angiogenic effect,was studied using NSC34 cells, a murine motor neuron cell line⁴⁰, inresponse to various apoptotic stimuli. Known motor neuron survivalfactors (bFGF⁴¹, TGFβ-1⁴²) protected NSC34 cells against TNF-α-inducedapoptosis (FIG. 5a). Physiological concentrations of VEGF₁₆₅ alsoprotected motor neurons against apoptosis induced by TNF-α, hypoxia,oxidative stress (H₂O₂), and serum deprivation. Notably, VEGF₁₂₁ (whichdoes not bind NP-1¹⁰) did not rescue motor neurons. Involvement of NP-1was demonstrated by the partial neutralization of the VEGF₁₆₅ survivaleffect by antibodies, blocking NP-1 but not by antibodies blocking NP-2.The neurotrophic effect of VEGF₁₆₅ was also partially blocked byantibodies to VEGFR-2 but not to VEGFR-1, while complete neutralizationwas achieved by the combination of both VEGR-2 and NP-1 antibodies. NP-1is known to bind semaphorin III/collapsin-1 (Sema3A), implicated inrepulsion and patterning of sensory and motor projections in the spinalcord during development¹¹⁻¹⁵ Sema3A was recently suggested to promoteapoptosis of sympathetic and cerebellar neurons⁴³, and to prevent axonalregeneration after nerve injury in the adult⁴⁴ (and, therefore, could beimplicated in axon retraction and motor neuron death in VEGF^(m/m)mice), Exposure of wild type mice to hypoxia (10% O₂; 24 h) slightlyincreased expression of Sema3A in the spinal cord. SCN34 motor neuronsalso expressed Sema3A. Thus, motor neurons express both aneuroprotective factor (VEGF₁₆₅) as well as a neurorepulsive/apoptoticfactor (Sema3A), that are reciprocal antagonists for binding to NP-1.

[0064] 10. Abnormal Neuronal Perfusion in VEGF^(m/m) Mice

[0065] We examined whether the muscle weakness and neuropathy werecaused by vascular insufficiency. In VEGF^(m/m) skeletal muscle, only areduction in capillary lumen size could be detected, but microvascularpartial oxygen pressure measurements revealed that the smallercapillaries did not cause muscle ischemia. Importantly, thecapillary-to-muscle ratio was normal when the first signs of neurogenicmuscle atrophy developed, showing that impaired angiogenesis was not thecause of motor neuron degeneration. Instead, the slight decrease of thisratio in old VEGF^(m/m) mice with severe muscle atrophy beyond 7 monthsmay be the result of muscle denervation, as observed in patients withdenervation muscle atrophy (Carpenter et a/. (1982) Muscle Nerve 5,250). Furthermore, PCNA-labeling failed to detect genotypic differencesin endothelial proliferation in skeletal muscle at all ages andfluoro-angiography (Schratzberger P. et al. (2000) Nat Med 6, 405) ofthe diaphragm revealed comparable vascularization in both genotypes. Noobvious structural vascular defects could be detected in neuronal tissuebut, surprisingly, neuronal perfusion was reduced by 50% in VEGF^(m/m)mice. In sciatic nerves, both genotypes had a comparable density of vasanervorum (capillaries/mm²: 90±6 in VEGF^(+/+) mice versus 100±7 inVEGF^(m/m) mice; n=7; p=NS) and pattern of peri- and endoneural vesselswithout signs of leakiness or obstruction (fluoro-angiography). In thespinal cord, capillary densities were comparable in both genotypes inthe gray matter (capillaries/mm²: 380±17 in VEGF^(+/+) mice versus390±13 in VEGF^(m/m) mice; n=7; p=NS) and in the white matter(capillaries/mm²: 170±12 in VEGF^(+/+) mice versus 170±11 in VEGF^(m/m)mice; n=7; p=NS), with similar densities in ventral and dorsal horns.Endothelial cells in VEGF^(m/m) mice expressed blood-brain barriercharacteristics (glucoseransporter type I; Glut-1), but ultrastructuralsigns of diabetic microangiopathy (Boulton A. J. et al. (1998) Med ClinNorth Am 82, 909) were not detected. Because of the inaccessibility andsmall size of the spinal cord, blood flow was quantified in the brainusing microspheres. Baseline cerebral blood flow was 0.9±0.1 ml/min/g inVEGF^(+/+) mice versus 0.5±0.1 in VEGF^(m/m) mice (n=8; p<0.05).

[0066] VEGF^(m/m) mice were, however, still able to increase theircerebral blood flow in reponse to hypercapnia (7.5% CO₂), as measuredusing laser doppler (% increase of flow: 43±3% in VEGF^(+/+) mice versus39±6% in VEGF^(m/m) mice; n=10; p=NS). The neuronal perfusion deficitappeared to be specific as renal perfusion was normal in VEGF^(m/m) mice(1.5±0.2 ml/min/g in VEGF^(+/+) mice versus 1.8±0.3 in VEGF^(m/m) mice;n=8; p=NS). It should be clear that characteristic signs of diabeticneuropathy (hyalinization of endoneural microvessels, thickening ofcapillary basement membrane, pericyte drop-out, lumen obstruction due toendothelial hyperplasia/hypertrophy, neovascularization, nerve infarct)were not detected in VEGF^(m/m) mice. Furthermore, the muscle weaknessin VEGF^(m/m) mice was not due to impaired oxygenation, nor to reducedlevels of the O₂-carrier hemoglobin (normal hematological profile). Inaddition, echocardiographic determination of the circumferential fibershortening (VCF, a measure of contractility) revealed that VEGF^(m/m)mice had normal cardiac function during baseline conditions (15±2 inVEGF^(+/+) mice versus 17±3 in VEGF^(m/m) mice; p=NS) and afterdobutamine-stress (27±6 in VEGF^(+/+) mice versus 26±6 in VEGF^(m/m)mice; p=NS). There was also no metabolic imbalance in mutant mice(normal electrolytes; plasma glucose levels: 200±10 mg/dl in VEGF^(+/+)mice versus 180±16 in VEGF^(m/m) mice, n=7; p=NS). In conclusion, themuscle weakness and neuropathy in VEGF^(m/m) mice were not due toreduced oxygen saturation levels in the blood, anemia, metabolicimbalance or cardiac dysfunction.

[0067] Materials and Methods

[0068] 1. Generation of VEGF^(m/m) Mice

[0069] The murine VEGF gene (129/SvJ; Genome Systems Inc., St. Louis,Mo.) was isolated and mapped previously²⁶. Deletion of the HIF-1alfabinding site in the VEGF promoter was achieved by constructing atargeting vector, pBSK.VEGF^(m), in which the wild type TACGTGGGHIF-1alfa response element (HRE) was deleted, which abolishes HIF-1alfabinding²³. This vector contained a neomycin phosphotransferase (neo)cassette, flanked by loxP sites to allow subsequent removal byCre-recombinase (FIG. 1a). After electroporation of pBSK.VEGF^(m),recombined ES cell clones, containing both the HIF-1alfa binding sitedeletion and the floxed neo-cassette (VEGF^(+/neo)) were identified bySouthern blot analysis and sequencing (FIG. 1a). VEGF^(neo/neo) ES cellswere obtained by culturing VEGF^(+/neo) ES cells in high G418 selection(1800 μg/ml), and used to obtain VEGF^(m/m) ES cells by transientexpression of the Cre recombinase. Probes for Southern blot analysisincluded: probe A (0.7 kb Pst1/BstEII fragment) and probe B (1 kb PCRfragment, amplified from genomic DNA, using as forward primer 5′-TTA TCAGM TTC ATT CCC GAG GCC TGG GGA GAG TTG GG-3′and as reverse primer 5′-ATAMG AAT TCG GAA GGT CAC AGC CCT TCG GTG G-3′). Analytical restrictiondigests used for identification of recombinant ES cell clones areindicated. Targeted VEGF^(+/neo) ES clones were used to generatechimeric mice via morula aggregation, that were testbred with Swissfemales for germline transmission. Viable VEGF^(+/neo) offspring werenot obtained, presumably because the presence of the neo-geneinactivated VEGF gene expression and caused haploinsufficient lethality.However, when chimeric mice were intercrossed with pgk:Cre mice, viableVEGF^(+/m) offspring were obtained, that were intercrossed to obtainhomozygous VEGF^(m/m) offspring. All methods of ES culture, selection,and diploid aggregation have been described²⁶.

[0070] 2. Gene Expression, Morphology, Motor Performance Tests, Torqueand Electromyography

[0071] Western and Northern blotting, quantitative real-time RT-PCR,histology, electron microscopy, immunostaining, alone or in combinationwith in situ hybridization, and morphometric analysis were performed aspreviously described^(4,26,56). The following antibodies were used forimmunostaining: Glut-1 (C-20; Santa Cruz Biotechnology Inc, Santa Cruz,Calif.), VEGF (Santa Cruz), desmin (D33; Dako S/A, Glostrup, Denmark),ChAT (AB144; Chemicon, Biognost, Wevelgem), NF (SM32; SternbergerMonoclonals Inc., Lutherville, Md.), NF_(P) (SMI 31; SternbergerMonoclonals Inc.), calretinin (Swant, Bellinzon, Switzerland), MAP2(Sigma, Bornem, Belgium), GFAP (Z0334; Dako S/A), ubiquitin (Z0458; DakoS/A), synaptophysin (A0010; Dako S/A), F4/80 (A3-1; Serotec Ltd, Oxford,UK), pimonidazole hydrochloride (Hypoxyprobe-1; Natural PharmaciaInternational Inc., Belmont, Md.), BrdU (Beckton Dickinson, Brussels,Belgium). Histochemical staining (myosin ATPase, Nissl, Bielschowski)was performed using standard protocols. All stainings were performed on7 μpm-thick sections, except for ChAT (40 μm), Nissl (15 μm) and myosineATPase (15 μm). Quantitative real-time RT-PCR analysis was performed aspreviously described⁵⁶. The relative expression levels of these geneswere calculated by dividing their signals by the signals obtained forthe HPRT gene.

[0072] Motor coordination and muscular performance tests (footprinttest, hanging test, grip test, rotating axle test)²⁹, electromyographicrecordings in anesthetized mice⁵⁸, and echocardiographic analysis⁵⁶ wereperformed as described. All animal procedures were approved by theethical committee. For fluoro-angiography⁶, 500 μl of 5% fluorescentdextran (molecular weight of 2×10⁶ dalton; Sigma) was injectedintravenously in urethane-anesthetized mice. After 5 minutes, mice wereperfused with 1.9 ml fluorescent dextran and 100 μl adenocor (SanofiPharma, Brussels, Belgium), and sciatic nerves were immediately analyzedby confocal microscopy. Laser-doppler measurements of blood flow (bloodperfusion units) through sciatic nerves was performed in anesthetizedmice using a needle flow probe (ADInstruments Pty Ltd, Castle Hill,Australia) at 1 mm intervals across a 5 mm nerve segment. Analysis ofblood gases, clinical chemistry and hematologic profile was performedusing standard techniques at the University Hospital (Leuven, Belgium).

[0073] 3. Cell Culture and Survival Analysis

[0074] SCN-34 cells were cultured as described⁴⁰. Blocking antibodies toNP-1 and NP-2 were a gift from Dr. A. Kolodkin, and VEGFR-2 antibodies(DC101) from Dr. P. Bohlen, (Imclone). For apoptosis studies, SCN-34cells were cultured in T75 flasks coated with 0.1% gelatin in RPMI 1640medium containing 10% foetal calf serum (Life Technologies, Paisley,UK), 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine,heparin (100 μg/ml) and endothelial cell growth supplement (30 μg/ml).Apoptosis was induced by supplementation of TNF-alfa (50 ng/ml; R&D,Abingdon, UK), withdrawal of growth factors (0.1% or 0.5% fetal calfserum), or treatment with hypoxia (2% O₂) or 2% H₂O₂. VEGF₁₂₁ andVEGF₁₆₅ were from R&D. Apoptosis was quantified by measuring cytoplasmichistone-associated DNA fragments (mono- and oligonucleosomes) using aphotometric enzyme immunoassay (Cell Detection ELISA, BoehringerMannheim, Mannheim, Germany). Determination of VEGF levels was performedusing commercially available ELISAs (R&D).

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1 2 1 38 DNA artificial sequence forward primer 1 ttatcagaat tcattcccgaggcctgggga gagttggg 38 2 34 DNA artificial sequence reverse primer 2ataaagaatt cggaaggtca cagcccttcg gtgg 34

1. Use of VEGF, or homologues, derivatives or fragments thereof, for themanufacture of a medicament to treat neuron disorders.
 2. Use accordingto claim 1, wherein said neuron disorders are neuronopathies.
 3. Useaccording to claim 2, wherein said neuronopathies are motor neurondisorders.
 4. Use according to claim 3 wherein said motor neurondisorders comprise amyotrophic lateral sclerosis and amyotrophic lateralsclerosis-like diseases.
 5. Use according to any of claims 1 to 4,wherein said VEGF is isoform VEGF_(165.)
 6. Use according to any claims1 to 4, wherein said use prevents death of motor neurons in the spinalcord.
 7. Use according to claim 1, wherein said neuron disorderscomprise Alzheimer's disease, Huntington disease, Parkinson's disease,chronic ischemic brain disease.
 8. Use of VEGF, or homologues,derivatives or fragments thereof, for the manufacture or a medicament toprevent and/or to treat cognitive dysfunction.
 9. Use of theneuropilin-1 receptor and VEGF Receptor-2 (VEGFR-2) to identifymolecules which stimulate survival of motor neurons or which inhibitsemaphorin 3A-induced death of neurons comprising: exposing theneuropilin-1 receptor and VEGFR-2 to at least one molecule whose abilityto modulate motor neuron survival is sought to be determined, andmonitoring survival of said motor neurons.
 10. A method for theproduction of a pharmaceutical composition comprising the usage of atleast one compound according to claim 9 and further more mixing saidcompound identified or a derivative or homologue thereof with apharmaceutically acceptable carrier.
 11. A non-human animal which ischaracterized by having an impaired or non-functional hypoxia-inducedVEGF expression compared to wild-type, non-human animals.
 12. Anon-human animal according to claim 11, wherein said animal is a rodent.13. Use of a non-human animal according to any of claim 11 and 12 toidentify and/or to test molecules to prevent and/or to treat neurondisorders or cognitive dysfuntion.
 14. Use of VEGF promoterpolymorphisms to identify individuals having a predisposition to acquirea neuron disorder and/or cognitive dysfunction.
 15. Use of HRE orpolymorphisms thereof for gene therapy of neuron disorders.